1. Field of the Invention
The present invention relates to a method of making dual specific antibodies. More specifically, the present invention relates to a method of making antibodies that are dual specific to both (1) amino acid sequences and (2) solid phase lipid structures. The present invention has relevance to such important subject matter as making broadly neutralizing monoclonal antibodies to HIV-1.
2. Brief Description of Related Art
One of the major barriers that have emerged in the development of an effective HIV-1 vaccine is the difficulty in obtaining neutralizing antibodies that block infection by primary isolates derived from a wide cross-section of clades (subtypes). In order to obtain broadly neutralizing or protective antibodies to HIV-1 it is necessary for antibodies to utilize antigenic epitopes (i.e., molecular recognition sites for binding of antibodies) that are conserved in the virus or that are present in the host or target cell in the regions in which the virus either buds or where binding or fusion with the virus occurs (McMichael & Hanke 2003; Burton et al. 2004). Most mammalian cells have a relatively conserved repertoire of lipids in the lipid bilayer of the plasma membrane, including glyceryl phospholipids, sphingosyl phospholipids (mainly sphingomyelin), lysophospholipids, glycosphingolipids, and cholesterol.
The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary isolates of virus have been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms.
It has been known for more than 20 years that monoclonal antibodies can have subsite specificities that simultaneously recognize different epitopes, such as simultaneous recognition of different types of carbohydrates; or combinations of carbohydrate and sulfated molecules, or carbohydrates and phosphorylated molecules. These subsites for different epitopes exist simultaneously in the same overall antigen binding site of the antibody. In our research, we have found polyclonal or monoclonal antibodies to membrane associated lipid antigens that also contain subsites that recognize unrelated phosphate or sulfated molecules as an epitope. We have also found that numerous membrane associated protein antigens have subsites that also recognize phosphate and even cross-react with phospholipids. However, this research has not produced a monoclonal antibody that is broadly neutralizing to HIV-1.
Therefore, an object of the present invention was to make antibodies that have dual specific action by recognizing, as antigens or epitopes, both (1) amino acid sequences such as proteins, peptides and polypeptides and also (2) solid phase lipid structures such as lipids, liposomes and the like so that the antibody will have greater affinity for these antigens or epitopes at the surface of target organisms or cells. The amino acid sequences and solid phase lipid structures may be from entities such as viruses, bacteria, cancer cells, hormones or any other substance that produces an immune response, wherein both (1) and (2) are capable of being recognized individually or together (i.e., simultaneously) by the antibody.
Another object of the invention was to apply this strategy to obtain antibodies that are broadly neutralizing to HIV-1 because they have subsites that recognize both protein and lipid or carbohydrate antigenic epitopes that are present either on the virus or on the budding site, receptor site, or fusion site of the plasma membrane.
In the case of HIV-1, this is necessary for the antibody to have dual specificity with the HIV-1 protein and with the plasma membrane of the host cell in the vicinity of the HIV-1 virus. In the case of other entities that produce an immune response, the antibodies will either be to the lipids themselves or to the combined lipid and amino acid sequences. The antibodies will either interfere with the entity through steric hindrance, or through conformational changes in the lipids that will interfere with the viability of the entity, or that will activate complement or other types of innate immunity as an effecter mechanism.
FIG. 4 is a schematic model of the HIV-1 putative trimeric envelope spike. The viral particle 2 is shown inserted into the plasma membrane 5. Most of the surface of gp 41 is believed to be occluded by gp120. However, the amino acid sequences of gp41 close to the membrane that have been identified as binding sites of MABs 2F5, Z13, and 4E10 have been suggested to be exposed to antibody binding (Zwick et al., 2001). IgG is shown as 20.
The invention solves the problems associated with the past lack of ability to find antibodies that are broadly neutralizing. In the case of HIV-1, the invention solves the problem by showing that patterns of plasma membrane lipids, known as lipid rafts, serve as binding sites not only for viral interactions with host and target cells, but also as lipids that might be incorporated into HIV-1 to comprise the lipid bilayer of the virus envelope and exploiting this knowledge to produce monoclonal or polyclonal antibodies that recognize these lipids as well as HIV-1 peptides. This invention will have particular relevance for HIV vaccine research and development, and for the treatment of HIV-1 and for research, vaccine development, and treatment of other enveloped viruses.